lncRNA SNHG1与miR-193a-5p相互作用调控胰腺癌发生发展的机制及诊断价值研究

Long noncoding RNAs (lncRNAs) play important regulatory roles in a variety of tumors. However, the functional involvement of these transcripts in pancreatic cancer are not thoroughly understood. Based on our preliminary data, SNHG1 is highly expressed in pancreatic cancer, and it promotes pancreatic cancer cell growth and invasion. Bioinformatic analysis revealed that SNHG1 may target miR-193a-5p, and KEGG pathway analysis showed that miR-193a-5p had potential target genes involved in the EGFR-Ras-Raf-MAPK signaling pathway. According to the preliminary results, we propose that SNHG1 contributes to pancreatic cancer tumorigenesis by acting as molecular sponge of miR-193a-5p, then activating the EGFR-Ras-Raf-MAPK pathway. We intend to elucidate the mechanism though analysis of clinical specimens, cell culture, tumor bearing nude mice and molecular biology experiments. First, we would like to explore the biological function of SNHG1 in pancreatic cancer. Next, we would confirm that SNHG1 regulates the expression of HER2 and KRAS through sponging miR-193a-5p, then activating the EGFR-Ras-Raf-MAPK pathway and promoting the development of pancreatic cancer. Finally, we would detect the expression level of SNHG1 in the serum of patients with primary pancreatic cancer (preoperative and postoperative) and recurrent tumor to provide novel diagnostic or prognostic biomarkers for pancreatic cancer.

lncRNA与肿瘤发生发展密切相关，但是其在胰腺癌中的作用还不确切。我们前期研究发现SNHG1在胰腺癌中高表达并促进细胞生长和侵袭，并且可能靶向miR-193a-5p发挥作用，应用KEGG通路分析发现miR-193a-5p与EGFR-Ras-Raf-MAPK通路密切相关，我们推断SNHG1通过吸附miR-193a-5p而调控EGFR-Ras-Raf-MAPK通路的活性，从而影响癌胰腺癌细胞生长和侵袭。本项目拟通过临床标本分析、细胞培养、裸鼠荷瘤实验及分子生物学实验，首先明确SNHG1在胰腺癌中的生物学功能；进而分析SNHG1通过miR-193a-5p调控HER2和KRAS的表达而激活EGFR-Ras-Raf-MAPK通路，促进胰腺癌发生发展的机制；最后通过检测胰腺癌患者术前、术后、复发病人血清中SNHG1的水平变化，评价其作为胰腺癌血清标志物的诊断价值，为胰腺癌的诊断治疗提供新的理论参考。

越来越多的研究表明，lncRNA与人类疾病包括肿瘤有密切联系。目前关于lncRNA与胰腺癌关系的研究还比较少，研究lncRNA在胰腺癌发病机制中的作用具有重要的意义。本研究发现lncRNA SNHG1在胰腺癌中显著上调，并且应用功能缺失和功能获得两方面的实验，发现SNHG1在胰腺癌细胞的增殖和侵袭中发挥关键调控作用。敲低SNHG1使胰腺癌细胞生长减慢，凋亡增加，并且侵袭能力降低；另一方面过表达SNHG1可以促进胰腺癌细胞生长和侵袭。这些结果表明，SNHG1可能作为癌基因发挥作用，其过表达有助于胰腺癌的进展。我们应用生物信息学软件预测到miR-193a-5p可以靶向SNHG1，RT-qPCR结果显示在胰腺癌组织和细胞中，miR-193a-5p的表达水平与SNHG1具有显著的负相关关系。过表达miR-193a-5p能够显著降低胰腺癌细胞内源性SNHG1的表达水平，提示miR-193a-5p能够负调控SNHG1的表达。本研究通过原位杂交和胞浆胞核分离实验，证实SNHG1主要定位于细胞浆，并且通过双荧光素酶报告基因实验和RIP实验发现miR-193a-5p能直接靶向SNHG1，并以Ago2依赖的方式调控SNHG1的表达。另外，SNHG1能通过miR-193a-5p调控EGFR-Ras-Raf-MAPK信号通路的活性。本研究揭示了miR-193a-5p、SNHG1及EGFR-Ras-Raf-MAPK信号通路的相互作用关系，从而将这两类重要的非编码RNA在胰腺癌中的作用联系起来，为阐明胰腺癌的发生发展机制提供了理论依据。最后，应用绝对定量RT-qPCR发现胰腺癌病人血清中SNHG1的含量及检出率显著高于正常对照人群，这为评价胰腺癌病人血清中lncRNA SNHG1能否作为胰腺癌的诊断及预后的分子标记提供了可行性。